Prolactin (PRL) is a peptide hormone from the anterior pituitary gland that participates in the regulation of a variety of targets. Its modus operandi is the subject of this research. The prevailing concept in this area that had previously guided experimental design was that peptide hormones, as "first messengers", could not enter their target cells and, therefore, that the generation of "second messengers", through an interaction of the hormone with the cell boundary, must be operative. In 1976, we demonstrated unequivocally not only that PRL can enter its major target, the milk secretory cell, but that it does so physiologically and that during its sojourn in the cell, it becomes intimately associated with the cell nucleus. Thus, an alternative to the "second messenger" hypothesis was born. The major objective of our present work is to continue to examine this alternative, namely, whether PRL regulation requires its direct contact with subcellular constituents. Our efforts during the proposed period of support would be directed along several major lines. The precise topogenesis of native, endogenous PRL in the normal rat milk secretory cell and the effects of the lactation-inhibitor, estrogen, on this process would be examined ultrastructurally. In vitro preparations of rat mammary gland would also be used to study the effects of a number of drugs known to modify uptake and intracellular transport. These, and the estrogen experiments, would be done to examine, in detail, the subcellular elements that might be involved in PRL topogenesis and the relationship of changes in intracellular PRL to cell response. To investigate mechanisms of action on the molecular level, PRL stimulation of subcellular fractions to form peptides with PRL-like bioactivity and the molecular relationship of such peptides to PRL and/or to the PRL receptor would be studied. End-point analysis of each of the experiments would utilize immunocytochemical electron microscopy (IEM) and would include concomitant IEM of the prolactin-dependent specific gene products: Alpha-lactalbumin, casein and thioesterase II to monitor cell responsiveness. Although this research constitutes a unique approach to resolving the most fundamental problem in biology, the regulation of genetic expression, its significance ipso facto is speculative at this moment. It is in retrospect that it must be evaluated.